From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium.
the Neuroscience Discovery Research, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, and.
J Biol Chem. 2011 Mar 11;286(10):8677-8687. doi: 10.1074/jbc.M110.194860. Epub 2011 Jan 5.
β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ∼0.76 nm), in primary neurons (EC(50) ∼1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ∼0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.
β-分泌酶（BACE1）是阿尔茨海默病的一个有吸引力的药物靶点。然而，设计针对其活性部位的临床有用抑制剂极具挑战性。为了确定替代药物靶向部位，我们生成了一组 BACE1 单克隆抗体（mAb），这些 mAb 在各种测定中干扰 BACE1 的活性，并确定了它们的结合表位。mAb 1A11 使用基于大 APP 序列的底物在体外抑制 BACE1（IC50∼0.76nm），在原代神经元（EC50∼1.8nm）中，以及在立体定向注射后在小鼠脑中抑制 BACE1。矛盾的是，当使用短合成肽作为底物时，mAb 1A11 增加了 BACE1 的体外活性，表明 mAb 1A11 不占据活性部位。表位作图显示，mAb 1A11 结合相邻的环 D 和 F，它们与附近的螺旋 A 一起，将 BACE1 与其他天冬氨酸蛋白酶区分开来。有趣的是，环 F 和螺旋 A 的突变降低或增加了 BACE1 的活性，将它们鉴定为酶调节元件和潜在的抑制剂设计替代位点。相比之下，mAb 5G7 是细胞游离酶测定中有效的 BACE1 抑制剂（IC50∼0.47nm），但在原代神经元中没有抑制作用。其表位是一个表面螺旋 299-312，在膜锚定的 BACE1 中不可用。值得注意的是，螺旋 299-312 的突变强烈降低了 BACE1 胞外结构域的脱落，表明该螺旋在 BACE1 的细胞生物学中起作用。总之，这项研究生成了高度选择性和有效的 BACE1 抑制性 mAb，这些 mAb 识别 BACE1 中的独特结构和功能元素，并揭示了 BACE1 上有趣的替代位点，这些位点可能成为药物开发的靶点。
